1. Field of the Invention
The invention comprises a rhamnogalacturonase (in the following usually abbreviated RGase), a corresponding DNA sequence, an RGase containing enzyme preparation and a use of the enzyme preparation.
Thus, the invention relates to genetic engineering and provides partial amino acid sequences of an RGase. These partial amino acid sequences can be used for construction of DNA probes which can be used for screening a genomic library from organisms expressing such enzyme, or a cDNA library, thereby obtaining DNA sequences, which can be used either for an overproduction of RGase, if inserted in the microorganism species, from which the parent DNA molecule originated, or for production of RGase without accompanying closely related enzymes, if inserted in a host microorganism, which in its not-transformed condition does not produce any enzymes closely related to RGase.
2. Description of Related Art
Plant cell walls comprising rhamnogalacturonans are of complex nature. Many publications deal with the polysaccharides serving as building blocks, of which these cell walls consist, and their importance with respect to the growing, ripening and processing of fruits and vegetables. Especially pectins have been studied frequently, because they are among the most important components in this respect. Pectins are proposed to consist of highly carboxyl-methylated linear homogalacturonan regions which alternate with "hairy" (ramified) regions that comprise highly branched rhamnogalacturonans mainly. Whereas the linear homogalacturonan regions are very well known and characterized, the structure of the so-called hairy regions is still not fully characterized, and thus is the subject of many investigations. But besides the scientific interest it is very important to be able to degrade these hairy regions for technical reasons. The enzymatic liquefaction of plant material like e.g. fruits, vegetables, cereals, oil fruits and seeds by technical processes involves combinations of pectolytic, cellulolytic and proteolytic enzyme preparations. This enzymatic treatment solubilizes the hairy regions and other pectic fragments, which originate from the insoluble cell wall protopectin. On one hand the solubilization of these polysaccharides is wanted, e.g. for the production of cloudy liquids and soluble dietary fiber containing solutions. On the other hand these polysaccharides cause problems during the processing of the clear liquids, because they are resistant to complete degradation of most technical enzyme preparations. Only one enzyme preparation (from Aspergillus aculeatus) has so far been described, which can degrade the rhamnogalacturonan backbone of the hairy regions. Therefore, it is of great importance for scientific (studies of the structures of these complex polysaccharides) and technical (liquefaction of plant material) reasons to obtain more knowledge about enzymes that can degrade these hairy regions. Especially for the industries dealing with modifications of plant cell walls for e.g. human nutrition and for animal feed (e.g. liquefaction of fruits, vegetables, cereals, oil fruits and seeds), it is of great importance to provide a great variety of different RGases (in respect to mode of action, pH and temperature range) in order to be able to exploit the desirable actions of RGases under widely varying technical process conditions.
RGase is described in the poster "Rhamnogalacturonase; a novel enzyme degrading the highly branched rhamnogalacturonan regions in apple pectic substances" from Wageningen Agricultural University, Department of Food Science, Biotechnion, Bomenweg 2, 6703 HD Wageningen, The Netherlands. From this poster is appears that a rhamnogalacturonase, the origin of which is not described, is well suited for degradation of the backbone of a modified "hairy region" (MHR) in plant cell walls. Also, it is described that this enzyme might play a role in the degradation of plant cell wall tissue, particularly in combination with other enzymes. However, it is not specified which other enzymes.
Also, the isolation and purification of RGase from Aspergillus aculeatus is described by Schols et al. in Carbohydrate Research 206 (1990) 105-115, "Rhamnogalacturonase: a novel enzyme, that degrades the hairy regions of pectins". From page 11, line 1 it appears that RGase has been purified to a high degree of purity as it moved as a single band in SDS-polyacrylamide gel electrophoresis.
Furthermore, in an article of Colquhoun in Carbohydrate Research 206 (1990) 131-144, "Identification by n.m.r. spectroscopy of oligosaccharides obtained by treatment of the hairy regions of apple pectin with rhamnogalacturonase" the composition of a mixture of oligosaccharides obtained by enzymatic degradation of the modified hairy (ramified) regions of apple pectin with a RGase is described.
The extraction of apple pectins by RGase is further described in the poster "Extraction of apple pectins by rhamnogalacturonase, a new pectolytic enzyme" by C. M. G. C. Renard et al., Laboratoire de Biochimie et Technologie des Glucides, INRA, Nantes (France).
To the best of applicant's knowledge, only a single species of RGase belongs to the prior art, and has been purified, i.e. the A. aculeatus RGase described by Schols et al., this RGase being the RGase appearing in all the previously indicated references. Also, this RGase has only been partly characterized and has not been characterized in regard to amino acid sequence, the corresponding RGase produced gene has not been cloned, and thus, the prior art RGase has not been available as a cheap, industrially useable product.
Thus, in consideration of what has been indicated previously, there is a great need for the provision of a variety of RGases with different properties corresponding to the conditions, under which rhamnogalacturonans have to be degraded industrially, e.g. different specificity, pH optimum and temperature optimum. Secondly, there is a need for cheap and pure RGases which can be used industrially in an economically sound manner.
Thus, the purpose of the invention is the provision of an RGase, which covers embodiments exhibiting varying characteristics corresponding to the different industrial conditions, under which rhamnogalacturonans have to be degraded, and of an RGase, which can be produced in better yield, and thus cheaper, than hitherto possible, and in higher purity. Also, it is the purpose of the invention to provide novel products, wherein the proportion of the RGase is either increased or decreased in relation to the proportion in the original product.